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Evaluation of the Hepatotoxic and Hepatoprotective Effect Of Evaluation of the hepatotoxic and hepatoprotective effect of Rwandese herbal drugs on (guinea pigs barbiturate-induced sleeping time) and (rat precision cut liver slices, PCLS) models Marie-Jeanne Mukazayire, V. Véronique Allaeys, Pedro Buc Calderon, Caroline Stévigny, Marie-Josée Bigendako, Pierre Duez To cite this version: Marie-Jeanne Mukazayire, V. Véronique Allaeys, Pedro Buc Calderon, Caroline Stévigny, Marie-Josée Bigendako, et al.. Evaluation of the hepatotoxic and hepatoprotective effect of Rwandese herbal drugs on (guinea pigs barbiturate-induced sleeping time) and (rat precision cut liver slices, PCLS) models. Experimental and Toxicologic Pathology, Elsevier, 2010, 62 (3), pp.289. 10.1016/j.etp.2009.04.005. hal-00592285 HAL Id: hal-00592285 https://hal.archives-ouvertes.fr/hal-00592285 Submitted on 12 May 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Author’s Accepted Manuscript Evaluation of the hepatotoxic and hepatoprotective effect of Rwandese herbal drugs on in vivo (guinea pigs barbiturate-induced sleeping time) and in vitro (rat precision cut liver slices, PCLS) models Marie-Jeanne Mukazayire, Véronique Allaeys, Pedro Buc Calderon, Caroline Stévigny, Marie-Josée Bigendako, Pierre Duez PII: S0940-2993(09)00171-7 www.elsevier.de/etp DOI: doi:10.1016/j.etp.2009.04.005 Reference: ETP50354 To appear in: Experimental and Toxicologic Pathology Received date: 14 July 2008 Revised date: 30 January 2009 Accepted date: 22 April 2009 Cite this article as: Marie-Jeanne Mukazayire, Véronique Allaeys, Pedro Buc Calderon, Caroline Stévigny, Marie-Josée Bigendako and Pierre Duez, Evaluation of the hepatotoxic and hepatoprotective effect of Rwandese herbal drugs on in vivo (guinea pigs barbiturate- induced sleeping time) and in vitro (rat precision cut liver slices, PCLS) models, Experi- mental and Toxicologic Pathology, doi:10.1016/j.etp.2009.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Evaluation of the hepatotoxic and hepatoprotective effect of Rwandese herbal drugs on in vivo (guinea pigs barbiturate-induced sleeping time) and in vitro (rat precision cut liver slices, PCLS) models Marie-Jeanne Mukazayire a,b ( ), Véronique Allaeys c, Pedro Buc Calderon c, Caroline Stévigny a, Marie-Josée Bigendako b, Pierre Duez a a. Laboratoire de Pharmacognosie, de Bromatologie et de Nutrition Humaine, Institut de Pharmacie, Université Libre de Bruxelles, CP 205/9, Bd du Triomphe, Bruxelles, Belgique. b. Centre de Recherches en Phytomédicaments et Sciences de la Vie, Institut de Recherche Scientifique et Technologique (I.R.S.T.), B.P. 227 Butare, Rwanda c. Unité de Pharmacocinétique, Métabolisme, Nutrition et Toxicologie, Faculté de Médecine, Université Catholique de Louvain, Bruxelles, Belgique Accepted manuscript ( ) Laboratoire de Pharmacognosie, de Bromatologie et de Nutrition humaine, Université Libre de Bruxelles (ULB), Campus de la Plaine – CP 205/9, Bd du Triomphe, B-1050 Bruxelles, Belgium. Phone, 32 - 2 - 650.5283; Fax, 32 - 2 - 650.5430; [email protected] 1 Abstract Precision-cut liver slices (PCLS) preserve the tissular organization of the organ and represent an in vitro model closer to in vivo conditions than hepatocytes cultures. As this may be an interesting tool for the investigation of hepatotoxic and protective effects but also for bioguided fractionations schemes, the usefulness of PCLS was compared with an in vivo test of liver function. Crude extracts derived from five herbs used in Rwanda for hepatoprotective activity were tested on CCl4-treated guinea pigs by the method of barbiturate-induced sleep modification. Ocimum lamiifolium, Crassocephalum vitellinum, Guizotia scabra and Vernonia lasiopus leaves extracts allowed animals to recover barbiturate sleep duration in proportions of 88, 78, 61 and 34 %, respectively and Microglossa pyrifolia was found inactive. Dried methanolic extracts of the 5 plants were then tested in vitro on rat PCLS for protection against acetaminophen - induced hepatotoxicity. In this model, Guizotia scabra, Microglossa pyrifolia and Vernonia lasiopus were found hepatotoxic by themselves and unable to prevent acetaminophen toxicity. The most active extract, obtained from Ocimum lamiifolium, was subjected to bioassay-guided fractionation by chromatography on Si-C18 to yield two quite active fractions. From a single animal, at least 50 PCLS explants can be prepared, which allows testing a large amounts of samples, strengthening ethnopharmacological data on hepatoprotective medicinal plants and investigating hepatotoxic effects. Accepted manuscript Key words: Rat precision-cut liver slices (PCLS); hepatotoxicity; hepatoprotection; Ocimum lamiifolium; Crassocephalum vitellinum; Guizotia scabra; Vernonia lasiopus; Microglossa pyrifolia; Guinea pigs; Sleeping time 2 1. Introduction The liver, major organ of metabolisation and excretion, is susceptible to a number of pathologies, classified as cirrhosis, acute chronic hepatitis and hepatitis; causes of hepatic trouble include parasitic and viral infections, autoimmune diseases and intoxication with various xenobiotics, such as chlorinated solvents, alcohol, drugs, herbal medicines, peroxidized fatty acids, fungal toxins, industrial pollutants and radioactive isotopes (Evans, 2002). The predominant pathologies in a given country depend on the lifestyle and economical conditions. In Rwanda, viral hepatitis and their complications, cirrhosis and hepatic carcinoma, represent 80 % of all liver pathologies, the 9th cause of morbidity (Musemakweli, 1999), and a number of plants are traditionally used to treat liver diseases (Van Puyvelde L., 1988). Excepting vaccines and interferon -2b, which concern only viral infections, modern medicine is quite limited in preventing or treating hepatic diseases; the only drugs available are cholagogues, choleretics, drugs for cholesterolic lithiasis, N-acetyl- cysteine and flavo-lignanes obtained from Silybum marianum. This limitation of therapeutic options gives considerable interest to the search for active compounds from plants traditionally used for these affections (Evans, 2002). In this context, an ethnopharmacological inquiry of a series of herbalists and/or traditional healers for plants used as remedies for liver diseases was undertaken by our team in Butare, Rwanda, in 2004, and confronted with published documents from Rwanda (Van Puyvelde L. et al., 1977; Van Puyvelde L., 1988; Rwangabo, 1993) and other countries (Demissew and N., 1994; Neuwinger, 2000). This led to Accepted manuscript the selection of five herbs locally used for hepatoprotective activity (Table 1), Crassocephalum vitellinum, Guizotia scabra, Microglossa pyrifolia, Ocimum lamiifolium and Vernonia lasiopus. Such ethnopharmacological reports however do not give information enough regarding the clinical efficaciousness or safety of herbs and there are risks possibly associated with uncontrolled/unsubstantiated use of herbal remedies. The present study is 3 intended to explore whether these herbs could have protective effect on hepatocytes and to give an orientation to find hepatoprotective compounds that may be present in the extracts; this first step should eventually lead us to the molecules responsible for the effect to investigate mechanisms of action on the liver. In order to investigate hepatotoxic and hepatoprotective compounds, i.e. agents capable to prevent and/or reverse the effects induced by an hepatotoxicant (CCl4, D-galactosamine, acetaminophen, peroxides,…), a series of models have been developed, either in vivo (histology, measurement of serum hepatic enzymes, barbiturate-induced sleeping time, prothrombine time, bromosulphaleine clearance) or in vitro (continuous cell lines, primary cultures of hepatocytes). Precision-cut liver slices (PCLS) preserve the tissular organization of the organ and represent an in vitro model closer to in vivo conditions than hepatocytes cultures (Morales et al., 1998; Vanhulle et al., 2001; Vickers and Fisher, 2004). As PCLS may be an interesting tool for the investigation of hepatotoxic and protective effects but also for bioguided fractionation schemes, their usefulness has been further investigated on the five ethnopharmacologically selected Rwandan herbal drugs, comparing to an in vivo test of liver function, barbiturate-induced sleeping time. 2. Material and methods: 2.1. Plant material The leaves of Crassocephalum vitellinum (Benth) S. Moore (Asteraceae), Guizotia scabra (Vis) Chiov (Asteraceae), Microglossa pyrifolia (Lam.) Kuntze (Asteraceae), Ocimum Accepted manuscript lamiifolium Hochst ex. Benth (Lamiaceae) and Vernonia lasiopus O. Hoffm (Asteraceae)
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